Excitotoxicity is defined as the deterioration of neuronal function/structure caused by excessive glutamatergic stimulation. It is a shared major pathological hallmark in many neurodegenerative diseases (ND), including Alzheimer?s disease (AD), Huntington?s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). Excitotoxicity is mostly mediated by the activation of the NMDA-type of glutamate receptors (NMDARs). However, the NMDAR function is indispensable for normal neuronal function. This conundrum is explained by the fact that NMDARs are segregated in two populations: synaptic (sNMDARs) and extrasynaptic (exNMDARs). While sNMDARs are linked to pro-survival signaling, over-activation of exNMDARs triggers excitotoxicity. Therefore, exNMDAR are obvious pharmacological targets in a broad range of ND and, in fact, blocking NMDAR activity strongly ameliorates cognitive defects in AD and HD mouse models. However, selective inhibition of exNMDARs is challenging, and the vast majority of NMDAR antagonists have failed in clinic due to side effects mediated by sNMDAR blockade. We propose to test a novel therapeutic strategy based on the fact that s- and exNMDARs are not independent populations. On the contrary, s- and exNMDARs pools are physiologically connected via lateral diffusion. We hypothesize that shifting the s/exNMDAR balance towards synaptic expression would be beneficial two-folds (i) promoting survival cascades (sNMDAR-mediated) and (ii) decreasing pro-death signaling (exNMDAR-mediated). We are ideally suited to test this strategy because we have previously identified several of the mechanisms controlling s/exNMDAR balance. Those include different protein interactions with the GluN2B-subunit of NMDARs and a particular phosphorylation on GluN2B (at S1480) that promotes sNMDAR clearance and receptor stabilization at extrasynaptic sites. The goal of this proposal is to validate the proof-of-principle that reducing excitotoxicity by preventing sNMDAR clearance and/or promoting exNMDAR reinsertion into synaptic sites is an effective therapeutic strategy in ND. In Aim 1, we will evaluate novel molecular tools to modulate s/exNMDAR balance in culture and in vivo, including (i) small interfering peptides (sIPs) and (ii) pharmacology to modulate GluN2B phosphorylation. Our study includes the repurposing of an anti-tumoral drug currently in phase 1/2 of clinical trial. Also, we will use proteomics to compare the posttranslational modification profile of s- vs. exNMDARs, aiming to identify novel mechanisms regulating the balance. In Aim 2, we will evaluate the suitability of this strategy as a common therapeutic strategy in ND. First, we will test the efficacy of our tools in ameliorating excitotoxicity-mediated pathological outcomes in several models of AD, both in culture and in vivo. Finally, we will use our strategy in primary cultures from models of HD (associated by excitotoxicity) and Parkinson?s disease (excitotoxicity is not a primary pathomechanism). If successful, this proposal, based on reducing excitotoxicity by regulating NMDAR trafficking but not by inhibiting NMDAR function, will have a groundbreaking translational impact on the identification of innovative therapeutics for a wide range of ND.